The invention relates to animals in which the MutS homolog 4 (MSH4) gene is misexpressed and methods of using such animals or cells derived therefrom, e.g., in methods of evaluating fertility treatments.
The DNA mismatch repair system (MMR) in mammalian cells is responsible for the repair of DNA mismatches that can result from a number of different mechanisms including DNA replication, genetic recombination and chemical modification of DNA or nucleotide pools. Studies in yeast, and more recently in mice, have also revealed a role for M proteins in the control of meiotic recombination. The bacterial DNA mismatch repair system typified by the E. coli Mut HLS system is the simplest and best understood. This system is capable of repairing both single nucleotide mismatches as well as small insertion/deletion mismatches (Kolodner, R. (1996) Genes Dev 10: 1433-1442, Modrich, P. et al. (1996) Annu Rev Biochem 65: 101-133). In E. coli, the Mut S protein recognizes and binds to mismatched nucleotides. In a subsequent step a second protein, Mut L, interacts with Mut S and activates a third protein, Mut H, which is an endonuclease. Mut H nicks the unmethylated strand of hemimethylated DNA in the vicinity of a mismatch, thereby directing the repair of the newly synthesized strand.
While the essential components of this MMR system have been conserved in eukaryotes, the repair system is more complex than in E. coli and involves several Mut S and Mut L homologs. In yeast Saccharomyces cerevisiae there are six homologs of the DNA binding protein Mut S designated Mut S homolog (MSH) 1-6. There are also four known homologs of the Mut L gene in yeast, designated MLH1, MLH2, PMS1 and MLH3 (Kolodner, R. (1996) Genes Dev 10: 1433-1442, Crouse, G. F. (1998) InDNA Repair in Prokaryotes and Lower Eukaryotes pp. 411-448). The mammalian genome has homologs for all of these genes except MSH1 which, if present, is yet to be discovered (Buermeyer, A. B., et al (1999) Annu. Rev. Genet. 33: 533-564, Kolodner, R. (1996) Genes Dev 10: 1433-1442).
It is well established that in eularyotes the products of the MSH2, MSH3, MSH6, as well as MLH1, PMS1 and MLH3 genes are involved in DNA mismatch repair. In eukaryotes, MMR requires a complex of MSH2-MSH6 for the repair of single base mispairs and either a complex of MSH2-MSH6 or MSH2-MSH3 for the repair of insertion/deletion mispairs (Acharya, S. et al. (1996) Proc Natl Acad Sci USA 93: 13629-13634, Marsischky, G. T. et al. (1996) Genes Dev 10: 407-420, Genschel, J. et al. (1998) J Biol Chem 273: 19895-19901, Guerrette, S., et al. (1998) Mol Cell Biol 18: 6616-6623, Umar, A. et al. (1998) Genetics 148: 1637-1646). The two MSH complexes interact with the complexes of MLH1-PMS1 (PMS2 in human) or MLH1-MLH3 for the repair of the different mismatches (Prolla, T. A. et al. (1998) Nat Genet 18: 276-279, Li, G. M. et al. (1995) Proc Natl Acad Sci USA 92: 1950-1954, Habraken, Y. et al. (1997) Curr Biol 7: 790-793, Pang, Q. et al. (1997) Mol Cell Biol 17: 4465-4473, Flores-Rozas, H. et al. (1998) Proc Natl Acad Sci USA 95: 12404-12409, Wang, T. F. et al. (1999) Proc Natl Acad Sci USA 96: 13914-13919).
Germ line mutations in some of the MMR genes in humans are associated with the cancer predisposition syndrome, hereditary non-polyposis colon cancer (HNPCC). This syndrome is inherited in an autosomal dominant fashion and is characterized by a predispostion to develop colonic and extracolonic tumors where the tumors have a characteristic replication error (RER+) phenotype (Kinzler, K. W. et al. (1996) Cell 87: 159-170). Germ-line mutations in MSH2 and MLH1 account for a majority of HNPCC families (Peltomaki, P. et al. (1997) Gastroenterology 113: 1146-1158). Recently it is was found that MSH6 germ-line mutations account for a small number of HNPCC families but appear to be also responsible for a larger number of late-onset familial colorectal cancer cases (Wu, Y. et al. (1999) Am J Hum Genet 65: 1291-1298).
Studies in bacteria and yeast showed that the MMR system is also involved in the control of recombination. For example, genetic analysis in yeast showed that the complexes consisting of the MMR proteins MSH2-MSH6, MSH2-MSH3, and MLH1-PMS1 function in the prevention of recombination between divergent DNA sequences. This role in recombination is dependent on interactions with other proteins including RAD1-RAD10 and EXO1 (Nakagawa, T. et al. (1999) Proc Natl Acad Sci USA 96: 14186-14188). Two other members of the yeast MSH family, MSH4 and MSH5, play a role specifically in meiotic recombination. Yeast strains carrying null mutations in either MSH4 or MSH5 show reduced rates of crossing over but not gene conversion, increased chromosomal nondisjunction and reduced spore viability (Ross-Macdonald, P. et al. (1994) Cell 79: 1069-1080, Hollingsworth, N. M. et al. (1995) Genes Dev 9: 1728-1739). The analysis of MSH4/MSH5 double mutant yeast strains indicates that MSH4 and MSH5 function in the same genetic pathway with MSH5 being epistatic to MSH4 (Hollingsworth, N. M. et al. (1995) Genes Dev 9: 1728-1739). Yeast MSH4 and MSH5 are able to form heterodimeric complexes similar to the mitotic MSH proteins (Pochart, P., D. et al. (1997) J Biol Chem 272: 30345-30349). In a manner analogous to mitotic MMR, the analysis of MSH4/MLH1 double mutant yeast strains indicated that the meiosis specific MutS homologs require the function of MLH1 for the promotion of meiotic crossing-over (Hunter, N. et al. (1997) Genes Dev 11: 1573-1582).
To understand the role of the mammalian mismatch repair genes in DNA repair, cancer predisposition and meiosis, several mouse lines with targeted mutations in MMR genes have been generated. Mice that carry mutations in the mismatch repair genes Msh2 (de Wind et al. (1995) Cell 82:321-330; Reitmair et al. (1995) Nat Genet 11:64-70), Msh3 (de Wind et al. (1999) Nat Genet 23:359-362; Edelmann et al. (2000) Cancer Res 60:803-807), Msh6 (Edelmann et al. (1997) Cell 91:467-477), Mlh1 (Baker et al. (1996) Nat Genet 13:336-342; Edelmann et al. (1996) Cell 85:1125-1134), Pms2 (Baker et al. (1995) Cell 82:309-319) and Pms1 (Prolla et al. (1998) Nat Genet 18:276-279) have been described. Msh2xe2x88x92/xe2x88x92, Mlh1xe2x88x92/xe2x88x92, Msh6xe2x88x92/xe2x88x92 and Pms2xe2x88x92/xe2x88x92 mice display a predisposition to tumors, although the degree of this predisposition and the latency for tumor development differ. Mice lacking Msh3 and Pms1 are reported to be normal.
Mice that are homozygous for mutations in the somatic members of the MSH gene family (Msh2, Msh3 and Msh6), are viable and fully fertile (de Wind et al. (1995) Cell 82:321-330; Reitmair et al. (1995) Nat Genet 11:64-70; Edelmann et al. (1997) Cell 25 91:467-477); Edelmann et al. (2000) Cancer Res 60:803-807). However, mice that are mutant for the mutL homologs Pms2 and Mlh1 also exhibit a meiotic defect in addition to their cancer predisposition phenotypes. Male mice bearing a homozygous mutation in Pms2 show abnormal chromosome pairing during meiosis and are sterile while the females are fertile (Baker et al. (1995) Cell 82:309-319). Mice with mutations in the Mlh1 gene are viable but both sexes are sterile. In spermatocytes from Mlh1 mutant males normal chromosome pairing was observed in pachynema of prophase I, but most of the cells fail to progress beyond pachynema (Baker et al. (1996) Nat Genet 13:336-342; Edelmann et al. (1996) Cell 85: 1125-1134).
The observation that mutations in the mutL homologous genes result in a different meiotic phenotype compared to mutations in the mutS homologous genes with which they interact during mitotic DNA mismatch repair indicates that the MLH proteins employ different members of the MSH family as partners during meiosis. Recently the human homologs of the yeast MSH4 and MSH5 genes have been isolated and their expression in human germ cells (Paquis-Flucklinger et al. (1997) Genomics 44:188-194; Her C. et al. (1998) Genomics 52:50-61; Winand et al. (1998) Genomics 53:69-80) suggests that one or both of these gene products may be partners for MLH1 during meiosis. Indeed Msh5 mutant mice are viable but both males and females are sterile. Meiosis in these mice cannot progress normally because chromosome pairing is severely affected during prophase I (de Vries et al. (1999) Genes Dev 13:523-531; Edelmann et al. (1999) Nat Genet 21:123-127).
The present invention is based, at least in part, on the generation of animals which are homozygous for a null mutation in the MutS homolog 4 (MSH4) gene and the observation that MSH4 is required for normal chromosome pairing during prophase I. The present invention is further based, at least in part, on the discovery that MSH4 and MSH5 are both essential for proper chromosome pairing during mammalian meiosis and that they act in the same pathway.
Accordingly, the invention features a non-human animal in which the gene encoding the MutS homolog 4 (MSH4) protein is misexpressed.
In preferred embodiments the animal, which is preferably a transgenic animal, is a mammal, e.g., a non human primate or a swine, e.g., a miniature swine, a monkey, a goat, or a rodent, e.g., a rat, but preferably a mouse.
In preferred embodiments, expression of the gene encoding the MSH4 protein is decreased as compared to the wild-type animal. For example, the levels of the MSH4 protein can be suppressed by, at least, 50%, 60%, 70%, 80%, 90%, or 100% as compared to the wild-type animal.
In preferred embodiments, misexpression of the gene encoding the MSH4 protein is caused by disruption of the MSH4 gene. For example, the MSH4 gene can be disrupted through removal of DNA encoding all or part of the protein.
In preferred embodiments, the animal can be heterozygous or homozygous for a misexpressed MSH4 gene, e.g., it can be a transgenic animal heterozygous or homozygous for an MSH4 transgene.
In preferred embodiments, the animal is a transgenic mouse with a transgenic disruption of the MSH4 gene, preferably an insertion or deletion, which inactivates the gene product.
In another aspect, the invention features a nucleic acid molecule which, when introduced into an animal or cell, results in the misexpression of the MSH4 gene in the animal or cell. In preferred embodiments, the nucleic acid molecule, includes an MSH4 nucleotide sequence which includes a disruption, e.g., an insertion or deletion, and preferably the insertion of a marker sequence. For example, a nucleic acid molecule can be the targeting construct shown in FIG. 2.
In another aspect, the invention features a method for identifying a compound that modulates, e.g., inhibits, the interaction between MSH4 and MSH5. The method includes contacting, e.g., directly or indirectly, MSH4 with the compound and determining the ability of the compound to modulate the interaction between MSH4 and MSH5.
In another aspect, the invention features a method for identifying a contraceptive compound. The method includes contacting, e.g., directly or indirectly, MSH4 with a test compound and determining the ability of the test compound to inhibit an activity of MSH4 e.g., the ability of MSH4 to interact with MSH5 or other molecules that function in the same genetic pathway as MSH4, thereby identifying a contraceptive compound.
In another aspect, the invention features a method for effecting contraception in a subject, e.g., a human, by administering to the subject a compound that inhibits an activity of MSH4, e.g., the ability of MSH4 to interact with MSH5 or other molecules that function in the same genetic pathway as MSH4.
In another aspect, the invention features a method for modulating, e.g., inhibiting, meiotic recombination in a cell by contacting the cell with a compound that modulates, e.g., inhibits, an activity of MSH4, such as the interaction between MSH4 and MSH5.
In another aspect, the invention features a method of evaluating a fertility treatment. The method includes administering the treatment to an MSH4 misexpressing animal, e.g., a transgenic mouse, or a cell derived therefrom, and determining the effect of the treatment on a fertility indication, e.g., sperm count, testicular size, or oocyte morphology, to thereby evaluate the treatment for fertility. The method may be performed in vivo or in vitro.
In preferred embodiments, the animal or cell is an animal or cell described herein. In other preferred embodiments, the method uses a transgenic mouse in which the expression of the MSH4 gene is inhibited. In yet other preferred embodiments, the method uses a cell derived from a transgenic mouse in which the expression of the MSH4 gene is inhibited.
In another aspect, the invention features a method for identifying a compound which modulates, e.g., inhibits, an activity of MSH4. The method includes contacting, e.g., directly or indirectly, MSH4 with a test compound and determining the effect of the test compound on an activity of MSH4 to, thereby, identify a compound which modulates an MSH4 activity. In preferred embodiments, the activity of MSH4 is inhibited.
In another aspect, the invention features a method for modulating the activity of MSH4. The method includes contacting, e.g., directly or indirectly, MSH4 or a cell expressing MSH4 with a compound which binds to MSH4 in an amount sufficient (e.g., a sufficient concentration) to modulate the activity of MSH4. In preferred embodiments, the activity of MSH4 is inhibited, e.g., the method can be used in contraception.
In another aspect, the invention features a method of identifying a subject having or at risk of developing a fertility disease or disorder. The method includes obtaining a sample from the subject; contacting the sample with a nucleic acid probe or primer which selectively hybridizes to MSH4 and determining whether aberrant MSH4 expression or activity exists in the sample, thereby, identifying a subject having or at risk of developing a fertility disease or disorder.
In another aspect, the invention features an isolated cell, or a purified preparation of cells, from an MSH4 misexpressing animal, e.g., an MSH4 misexpressing animal described herein. In preferred embodiments, the cell is a transgenic cell, in which the gene encoding the MSH4 protein is misexpressed. The cell, preferably a transgenic cell, can be an oocyte or a spermatocyte. In preferred embodiments, the cell is heterozygous or homozygous for the transgenic mutant gene.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.